Hippocampal slice cultures were transfected with eYFP using a gene-gun and processed for laser-scanning confocal microscopy. Quantitative analyses of dendritic spine density and geometrical measurements of their dimensions for spine type categorization were performed in individually identified and selected CA1 pyramidal neurons. A, left. NeuN immunocytochemistry was performed to identify hippocampal cytoarchitecture and subregions (i.e. DG, dentate gyrus; CA3 and CA1). The white box shows the area CA1 used to sample eYFP-expressing pyramidal neurons. middle. Representative CA1 pyramidal neuron showing the apical dendritic tree used for quantitative analyses of dendritic spines. right. Apical dendritic segment representative of those selected for confocal imaging; individual dendritic spines are marked with arrows. B, Classification of spine morphological types is based on the spine length, the maximum diameter of the spine head, and the maximum width of the spine neck. For the purposes of this study, three different types of dendritic spines were defined: stubby (type-I), mushroom (type-II) and thin (type-III) spines, to be consistent with our prior studies (see text for details).

A. Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-free media (SF) and treated with 250ng/mL BDNF for 48hrs. In this and all subsequent figures, scale bar represents 2µm. B. Dendritic spine density expressed per 10µm of apical secondary or tertiary dendrite. C. Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. D. Representative examples of dendritic segments of CA1 pyramidal neurons maintained in serum-containing media (SM) and treated with 250ng/mL BDNF for 48hrs. E. Dendritic spine density expressed per 10µm of apical secondary or tertiary dendrite. F. Proportion of each morphological type of dendritic spine, expressed as a fraction of the total spine population. In this and all subsequent figures, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001, after unpaired Student’s t test (see text for details).

A. Representative examples of immunostaining with anti-p75NTR and anti-TrkB antibodies from the apical dendritic region of area CA1 of hippocampal slice cultures followed by image deconvolution microscopy (scale bar represents 10µm). The arrows in the left panels show co-localization with profiles immunolabeled with antibodies against the postsynaptic protein PSD-95, indicating a synaptic localization of both BDNF receptors. B. Representative examples of Western immunoblots of whole-tissue homogenates prepared from hippocampal slice cultures maintained in serum-free or serum-containing media using anti-p75NTR and anti-TrkB antibodies. Gels were re-probed using antiactin antibodies as loading controls and for normalization of quantitative densitometric analyses.